Progress in understanding complex biological systems depends on characterizing the underlying interactions of biomolecules, in particular proteins. While the DNA sequencing of an increasing number of organisms has identified their open reading frames (ORF), the possibilities to characterize the corresponding proteins in vivo and in vitro are limited. Most strategies that aim at realizing this objective are based on the construction of a fusion protein that either allows the purification of the fusion for in vitro applications or allows following the protein in vivo. Examples for such tags include the 6xHis tag, glutathione S transferase, maltose binding protein, epitope tags, yeast-two hybrid system, O6-alkylguanine-DNA alkyltransferase, split-ubiquitin, and green fluorescent protein (GFP) fusion proteins. However, all these techniques have various limitations or disadvantages.
Gehring et al. (1997) and Lambalot and Walsh (1995) describe the use of E. coli holo acyl carrier protein synthase (ACPS) to catalyze the posttranslational modification of apo-acyl carrier protein (apo-ACP) by attaching the cofactor 4′-phosphopantetheine (P-pant) to a conserved serine residue in vitro, yielding holo-ACP. The source of P-pant is coenzyme A. Gehring et al. (1997) demonstrate furthermore that, by using coenzyme A analogs, which are modified in the P-pant part but still able to serve as substrates for ACPS, holo-ACP's with modified P-pants as cofactor are obtained.
Isolated phosphopantetheinyl transferases such as ACPS are described in International Patent Application WO 97/13845.
A method of transferring a label to O6-alkylguanine-DNA alkyltransferase (AGT) fusion proteins, and the use of this method for the detection of AGT fusion proteins is described in International Patent Application WO 02/083937.